Publisher Summary This chapter discusses the fundamental molecular properties of avidin and streptavidin. Carbohydrate-free avidin can be obtained by use of deglycosylating enzymes and it has been shown to be present in significant amounts in some commercial preparations from which it may be separated by use of lectin columns. Improvement on the original use of biotinyl cellulose came with the introduction of iminobiotinyl derivatives of Sepharose that utilized the pH dependence of the binding 24 to achieve efficient elution. In iminobiotin, the ureido group becomes a guanidinium group; the form in which this is uncharged is strongly bound. The gene for streptavidin has been cloned and sequenced with the ultimate objective of using it in general expression systems for detecting and isolating fusion proteins. The stability is greatly enhanced by biotin binding, because the total free energy of binding is about 330 kJ/mol of tetramer. The dissociation constant for biotin is so low that it can be estimated only from the ratio of the rate constants for binding and exchange. The binding is accompanied by a red shift of the tryptophan spectrum and by a decrease in fluorescence, either of which can be used as the basis for quantitative assays.

Avidin and streptavidin.

Chicken avidin and bacterial streptavidin, (strept)avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)avidin-biotin technology relies on the extremely tight and specific affinity between (strept)avidin and biotin (dissociation constant, Kd≈10−14–10−16 M). (Strept)avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)avidins, but no comprehensive reviews exist concerning genetically engineered (strept)avidins. To fill this gap, we here go through the genetically engineered (strept)avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)avidin-biotin technology.

Genetically engineered avidins and streptavidins.

Transport of ascorbic acid and other water-soluble vitamins.

Application of avidin-biotin technology to affinity-based separations

A method of egg yolk fractionation. Characterization of fractions

1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.

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Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin

Biotin-binding protein from egg yolk. A protein distinct from egg white avidin.

Preparation of lipid-free protein extracts of egg yolk

Publisher Summary This chapter describes the biotin-binding protein present in egg yolk. The assay is based on the temperature-accelerated exchange of bound biotin for free [ 14 C]biotin. The procedure is, therefore, useful for biotin-binding proteins with no free binding sites. The purification procedure for biotin-binding protein of egg yolk is described in the chapter. All buffers used are potassium phosphate, pH 7.2, at the concentration given. The procedure is for 5 dozen eggs. The eggs are cracked and the white and yolks are separated. The yolks are broken and 1.5 times the yolk volume in distilled water is added. The diluted yolk is well stirred. This solution is centrifuged at 13,200 g for 30 minutes. The supernatant is collected, and solid NaCl is added to make the solution 1 M in salt. After the salt has completely dissolved, the solution is poured into centrifuge bottles, two-third the solution volume in 1-butanol is added, and the mixture is shaken vigorously. The centrifugation of the resulting emulsion at 13,200 g for 15 minutes causes a lipid-free aqueous phase and a lipid-rich organic phase to form, with a solid layer of precipitated protein at the interface. The chapter discusses the properties of the biotin-binding protein.

Harry W. Meslar

Papers

Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin

Biotin-binding protein from egg yolk. A protein distinct from egg white avidin.

Preparation of lipid-free protein extracts of egg yolk

Egg yolk biotin-binding protein: assay and purification.